Explore the vast range of titles available.

Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. eaar2555 , p. eaar3958 , p. 412 ; see also p. 329 Phase-separated condensates compartmentalize the transcription apparatus at super-enhancers of key cell-identity genes. Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.

Structural view of a dynamic molecular switch mechanism that governs repression and activation of the jasmonate plant hormone pathway. Jasmonate hormone signalling The signalling pathway triggered by the plant hormone jasmonate regulates plant stress responses and also growth and development. At a molecular level, the jasmonate ZIM-domain (JAZ) proteins act as jasmonate co-receptors but also repress the activity of MYC transcription factors, which are required to convey the jasmonate signal. Sheng Yang He and colleagues use X-ray crystallography to address the question of how these same proteins can switch roles between transcriptional repressors and jasmonate co-receptors. Previous work had suggested that the conserved Jas motif of a JAZ repressor binds to jasmonate as a partially unwound helix. These authors find that the motif forms a complete α-helix on binding to MYC. Consequently, the Jas motif becomes an integral part of the MYC N-terminal fold resulting in a notable conformational change in MYC. This competitive binding inhibits MYC interaction with a subunit of the transcriptional Mediator complex, repressing its transcriptional activity. The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development 1 , 2 , 3 , 4 , 5 , 6 , 7 . Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase 8 , 9 , 10 , 11 . The hormone-dependent formation of the COI1–JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression 3 , 10 , 12 . The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.

Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt–Ada–Gcn5–acetyltransferase) 1 , 2 and transcription factor IID (TFIID) 2 – 4 . SAGA is required for all regulated transcription 5 and is conserved among eukaryotes 6 . SAGA contains four modules 7 – 9 : the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures 10 – 14 , but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6–Taf9, Taf10–Spt7 and Taf12–Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP) 2 , 7 , 15 – 17 . The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome. Structural studies on the yeast transcription coactivator complex SAGA (Spt–Ada–Gcn5–acetyltransferase) provide insights into the mechanism of initiation of regulated transcription by this multiprotein complex, which is conserved among eukaryotes.

CpG islands and chromatin modification Most human gene promoters are embedded within CpG islands that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. Here, a zinc-finger protein Cfp1 is found to be associated with non-methylated CpG islands and sites of H3K4me3 genome-wide in vivo . Cfp1 is part of the Setd1 complex which trimethylates H3K4. Artificial CpG clusters are shown to recruit Cfp1, leading to novel peaks of H3K4me3. Therefore a primary function of non-methylated CpG islands might be to genetically determine the local chromatin modification state. Most human gene promoters are embedded within CpG islands that lack DNA methylation and coincide with sites at which histone H3 lysine 4 is trimethylated (H3K4me3 sites). Here, a zinc-finger protein, Cfp1, is found to be associated with non-methylated CpG islands and H3K4me3 sites throughout the genome in the mouse brain. A primary function of non-methylated CpG islands might be to genetically determine the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins. CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides 1 , 2 . Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity 3 , 4 . In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro 5 , 6 . Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo . High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7 , 8 ). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.

Arabidopsis thaliana plants fend off insect attack by constitutive and inducible production of toxic metabolites, such as glucosinolates (GSs). A triple mutant lacking MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that are known to additively control jasmonate-related defense responses, was shown to have a highly reduced expression of GS biosynthesis genes. The myc2 myc3 myc4 (myc234) triple mutant was almost completely devoid of GS and was extremely susceptible to the generalist herbivore Spodoptera littoralis. On the contrary, the specialist Pieris brassicae was unaffected by the presence of GS and preferred to feed on wild-type plants. In addition, lack of GS in myc234 drastically modified S. littoralis feeding behavior. Surprisingly, the expression of MYB factors known to regulate GS biosynthesis genes was not altered in myc234, suggesting that MYC2/MYC3/MYC4 are necessary for direct transcriptional activation of GS biosynthesis genes. To support this, chromatin immunoprecipitation analysis showed that MYC2 binds directly to the promoter of several GS biosynthesis genes in vivo. Furthermore, yeast two-hybrid and pull-down experiments indicated that MYC2/MYC3/MYC4 interact directly with GS-related MYBs. This specific MYC—MYB interaction plays a crucial role in the regulation of defense secondary metabolite production and underlines the importance of GS in shaping plant interactions with adapted and nonadapted herbivores.

RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3–SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.

Key Points Cyclic AMP-responsive element-binding protein (CREB) mediates induction of cAMP-responsive genes following its phosphorylation at Ser133 by protein kinase A (PKA). CREB phosphorylation increases its activity by promoting an association with the co-activator paralogues CREB-binding protein (CBP) and p300. The cAMP-regulated transcriptional co-activators (CRTCs) mediate CREB target gene activation following their dephosphorylation and nuclear translocation, when they bind to CREB over relevant promoters. CRTCs are selectively activated by cAMP and calcium signals, perhaps explaining why only a subset of stimuli that promote CREB phosphorylation also increase target gene expression. CRTC1 is expressed almost exclusively in the hypothalamus, where it mediates effects of leptin on satiety. CRTC1 reduces food intake by stimulating the expression of the neuropeptide cocaine- and amphetamine-regulated transcript 1 (CART1) in arcuate cells. CRTC2 mediates effects of glucagon on induction of the gluconeogenic programme in the liver during fasting. CREB and CRTC2 activities are increased in insulin resistance, in which they contribute to the attendant hyperglycaemia. CRTC3 is expressed in white and brown adipose tissue, where it promotes obesity by inhibiting catecholamine signalling. Inheritance of a gain-of-function CRTC3 mutant in certain human populations is associated with obesity. The CRTCs are conserved in lower organisms, including Drosophila melanogaster and Caenorhabditis elegans , in which they mediate effects of fasting and feeding signals on glucose and lipid metabolism, as well as lifespan. The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, and it functions in concert with cAMP-regulated transcriptional co-activators (CRTCs). CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues. The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator–co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

Mutant p53 can attenuate the function of wild-type p53, p63 and p73. An aggregation-nucleating sequence in p53 that is revealed in structurally destabilized mutants can induce coaggregation with p63 and p73, resulting in their sequestration in cellular inclusions. Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ’s AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP–complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.